Methods and compositions for increasing red blood cells

ABSTRACT

In certain aspects, the present invention provides compositions and methods for increasing red blood cell and/or hemoglobin levels in vertebrates, including rodents and primates, and particularly in humans.

RELATED APPLICATION

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 61/621,154, filed Apr. 6, 2012. The specificationof the foregoing application is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

The mature red blood cell, or erythrocyte, is responsible for oxygentransport in the circulatory systems of vertebrates. Red blood cellscontain high concentrations of hemoglobin, a protein that binds oxygenin the lungs at relatively high partial pressure of oxygen (pO₂) anddelivers oxygen to areas of the body with a relatively low pO₂.

Mature red blood cells are produced from pluripotent hematopoietic stemcells in a process termed erythropoiesis. Postnatal erythropoiesisoccurs primarily in the bone marrow and in the red pulp of the spleen.The coordinated action of various signaling pathways control the balanceof cell proliferation, differentiation, survival and death. Under normalconditions, red blood cells are produced at a rate that maintains aconstant red cell mass in the body, and production may increase ordecrease in response to various stimuli, including increased ordecreased oxygen tension or tissue demand. The process of erythropoiesisbegins with the formation of lineage committed precursor cells andproceeds through a series of distinct precursor cell types. The finalstages of erythropoiesis occur as reticulocytes are released into thebloodstream and lose their mitochondria and ribosomes while assuming themorphology of mature red blood cell. An elevated level of reticulocytes,or an elevated reticulocyte:erythrocyte ratio, in the blood isindicative of increased red blood cell production rates.

Erythropoietin (EPO) is widely recognized as the most significantpositive regulator of postnatal erythropoiesis in vertebrates. EPOregulates the compensatory erythropoietic response to reduced tissueoxygen tension (hypoxia) and low red blood cell levels or low hemoglobinlevels. In humans, elevated EPO levels promote red blood cell formationby stimulating the generation of erythroid progenitors in the bonemarrow and spleen. In the mouse, EPO enhances erythropoiesis primarilyin the spleen.

Anemia is a broadly-defined condition characterized by lower than normallevels of hemoglobin or red blood cells in the blood. In some instances,anemia is caused by a primary disorder in the production or survival ofred blood cells. More commonly, anemia is secondary to diseases of othersystems (Weatherall & Provan (2000) Lancet 355, 1169-1175). Anemia mayresult from a reduced rate of production or increased rate ofdestruction of red blood cells or by loss of red blood cells due tobleeding. Anemia may result from a variety of disorders that include,for example, chronic renal failure, chemotherapy treatment,myelodysplastic syndrome, rheumatoid arthritis, and bone marrowtransplantation.

Treatment with EPO typically causes a rise in hemoglobins by about 1-3g/dL in healthy humans over a period of weeks. When administered toanemic individuals, this treatment regimen often provides substantialincreases in hemoglobin and red blood cell levels and leads toimprovements in quality of life and prolonged survival. EPO is notuniformly effective, and many individuals are refractory to even highdoses (Horl et al. (2000) Nephrol Dial Transplant 15, 43-50). Over 50%of patients with cancer have an inadequate response to EPO,approximately 10% with end-stage renal disease are hyporesponsive(Glaspy et al. (1997) J Clin Oncol 15, 1218-1234; Demetri et al. (1998)J Clin Oncol 16, 3412-3425), and less than 10% with myelodysplasticsyndrome respond favorably (Estey (2003) Curr Opin Hematol 10, 60-67).Several factors, including inflammation, iron and vitamin deficiency,inadequate dialysis, aluminum toxicity, and hyperparathyroidism maypredict a poor therapeutic response. The molecular mechanisms ofresistance to EPO are as yet unclear. Recent evidence suggests thathigher doses of EPO may be associated with an increased risk ofcardiovascular morbidity, tumor growth, and mortality in some patientpopulations (Krapf et al., 2009, Clin J Am Soc Nephrol 4:470-480;Glaspy, 2009, Annu Rev Med 60:181-192). It has therefore beenrecommended that EPO-based therapeutic compounds(erythropoietin-stimulating agents, ESAs) be administered at the lowestdose sufficient to avoid the need for red blood cell transfusions(Jelkmann et al., 2008, Crit Rev Oncol. Hematol 67:39-61).

Thus, it is an object of the present disclosure to provide alternativemethods and compositions for increasing red blood cell levels inpatients.

SUMMARY OF THE INVENTION

In part, the disclosure demonstrates that BMP9 polypeptides or BMP10polypeptides may be used to increase red blood cell and hemoglobinlevels. In particular, the disclosure demonstrates that BMP9, whenadministered in vivo, causes a profound and rapid increase in red bloodcell levels, hematocrit and hemoglobin. BMP10 is closely related to BMP9and is known to signal through the same set of receptors. Therefore, incertain embodiments, the disclosure provides methods for using BMP9 orBMP10 polypeptides (or a combination thereof) to increase red blood celland hemoglobin levels in patients and to treat disorders associated withlow red blood cell or hemoglobin levels in patients in need thereof.

In certain aspects, the present disclosure provides BMP9 polypeptides.In certain embodiments, a BMP9 polypeptide has an amino acid sequencethat comprises, consists of, or consists essentially of, the amino acidsequence of SEQ ID NO: 1, 2, 3, 7, 8 or 16, or an amino acid sequencethat is at least 63%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to any of the foregoing. A BMP9 polypeptide may comprise anamino acid sequence that is encoded by a nucleic acid of SEQ ID NO:11,including any portion thereof, such as nucleotides 1121-1450 that encodethe mature portion of BMP9, and a BMP9 polypeptide may be encoded by anucleic acid that hybridizes to a nucleic acid that is complementary tothe sequence of nucleotides 1121-1450 of SEQ ID NO:11 under lessstringent, moderately stringent or highly stringent hybridizationconditions.

In certain aspects, the present disclosure provides BMP10 polypeptides.In certain embodiments, a BMP10 polypeptide has an amino acid sequencethat comprises, consists of, or consists essentially of, the amino acidsequence of SEQ ID NO: 4, 5, 6, 9, 10 or 17, or an amino acid sequencethat is at least 63%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to any of the foregoing. A BMP10 polypeptide may comprise anamino acid sequence that is encoded by a nucleic acid of SEQ ID NO:12,including any portion thereof, such as nucleotides 1108-1431 that encodethe mature portion of BMP10, and a BMP10 polypeptide may be encoded by anucleic that hybridizes to a nucleic acid that is complementary to thesequence of nucleotides 1108-1431 of SEQ ID NO:12 under less stringent,moderately stringent or highly stringent hybridization conditions.

In certain aspects, the disclosure provides pharmaceutical preparationscomprising a BMP9 or BMP10 polypeptide and a pharmaceutically acceptablecarrier. The BMP9 or BMP10 polypeptide may bind to one or more type I(e.g., ALK1, ALK2) or type II (e.g., ActRIIA, ActRIIB, BMPRII) receptorswith a Kd less than 10 micromolar, less than 1 micromolar, less than 100nanomolar, less than 10 nanomolar, or less than 1 nanomolar. Typically,a BMP9 or BMP10 polypeptide will bind to both a type I receptor and atype II receptor, although binding to one of the receptors may be at avery weak affinity. Optionally, the BMP9 or BMP10 polypeptide willstimulate expression from a SMAD1- or SMAD5-responsive promoter in acell, such as a promoter containing the BMP-responsive element (BRE)from the ID1 gene.

A pharmaceutical preparation may further comprise a BMP9 prodomainpolypeptide or a BMP10 prodomain polypeptide. In certain embodiments, aBMP9 prodomain polypeptide has an amino acid sequence that comprises,consists of, or consists essentially of, the amino acid sequence of23-319 of SEQ ID NO: 1 or an amino acid sequence that is at least 63%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to same. A BMP9prodomain polypeptide may comprise an amino acid sequence that isencoded by the sequence of nucleotides 230-1120 of SEQ ID NO:11,including any portion thereof, and a BMP9 prodomain polypeptide may beencoded by a nucleic acid that hybridizes to a nucleic acid that iscomplementary to the sequence of nucleotides 230-1120 of SEQ ID NO:11under less stringent, moderately stringent or highly stringenthybridization conditions. In certain embodiments, a BMP10 prodomainpolypeptide has an amino acid sequence that comprises, consists of, orconsists essentially of, the amino acid sequence of 22-316 of SEQ ID NO:4 or an amino acid sequence that is at least 63%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% identical to same. A BMP10 prodomainpolypeptide may comprise an amino acid sequence that is encoded by thesequence of nucleotides 223-1107 of SEQ ID NO:12, including any portionthereof, and a BMP10 prodomain polypeptide may be encoded by a nucleicacid that hybridizes to a nucleic acid that is complementary to thesequence of nucleotides 223-1107 of SEQ ID NO:12 under less stringent,moderately stringent or highly stringent hybridization conditions. Aprodomain polypeptide may be covalently or non-covalently associatedwith a BMP9 or BMP10 polypeptide.

Preferably, a pharmaceutical preparation is substantially pyrogen free.In general, it is preferable that a BMP9 or BMP10 polypeptide beexpressed in a mammalian cell line that mediates suitably naturalglycosylation so as to diminish the likelihood of an unfavorable immuneresponse in a patient. Human and CHO cell lines have been usedsuccessfully, and it is expected that other common mammalian expressionvectors will be useful.

In certain aspects, the disclosure provides methods for making a BMP9 orBMP10 polypeptide. Such a method may include expressing any of thenucleic acids (e.g., SEQ ID NO: 11 or 12) disclosed herein in a suitablecell, such as a Chinese hamster ovary (CHO) cell. Such a method maycomprise: a) culturing a cell under conditions suitable for expressionof the BMP9 or BMP10 polypeptide, wherein said cell is transformed witha BMP9 or BMP10 expression construct; and b) recovering the BMP9 orBMP10 polypeptide so expressed. BMP9 or BMP10 polypeptides may berecovered as crude, partially purified or highly purified fractionsusing any of the well known techniques for obtaining protein from cellcultures. Purification may be achieved by contacting the BMP9 or BMP10polypeptide with a ligand binding domain of a receptor protein, such asALK1, ALK2, ActrIIA, ActRIIB or BMPRII or modified version thereof thatbinds to BMP9 or BMP10. The ligand binding domain may, for example, beused as a fusion with an Fc portion of an IgG (optionally with anintervening linker) and immobilized on a protein A-coated surface.

In certain aspects, a BMP9 or BMP10 polypeptide, or a pharmaceuticalpreparation comprising one or more of the foregoing, may be used in amethod for promoting red blood cell production or increasing red bloodcell levels in a subject. In certain embodiments, the disclosureprovides methods for treating a disorder associated with low red bloodcell counts or low hemoglobin levels (e.g., an anemia), or to promotered blood cell production, in patients in need thereof. A method maycomprise administering to a subject in need thereof an effective amountof a BMP9 or BMP10 polypeptide. In certain aspects, the disclosureprovides uses of BMP9 or BMP10 polypeptides for making a medicament forthe treatment of a disorder or condition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a multiple sequence alignment of human, murine and chickenBMP9 proteins. The alignment was obtained using the Clustal W program.

FIG. 2 shows a multiple sequence alignment of human, murine and chickenBMP10 proteins. The alignment was obtained using the Clustal W program.

FIG. 3 shows an alignment of the mature portions of BMP9 and BMP10.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. By manipulating theactivity of a member of the TGF-beta family, it is often possible tocause significant physiological changes in an organism. For example, thePiedmontese and Belgian Blue cattle breeds carry a loss-of-functionmutation in the GDF8 (also called myostatin) gene that causes a markedincrease in muscle mass. Grobet et al., Nat Genet. 1997, 17(1):71-4.Furthermore, in humans, inactive alleles of GDF8 are associated withincreased muscle mass and, reportedly, exceptional strength. Schuelke etal., N Engl J Med 2004, 350:2682-8.

Bone morphogenetic protein 9 (BMP9) and BMP10 are two closely relatedmembers of the TGF-beta superfamily. These proteins are thought to beproduced as disulfide linked homodimers that can circulate in the blood.BMP signals are mediated by heteromeric complexes of type I and type IIserine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massague, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors aretransmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling. Type II receptors are requiredfor binding ligands and for expression of Type I receptors. Type I andII activin receptors form a stable complex after ligand binding,resulting in phosphorylation of Type I receptors by Type II receptors.BMP9 and BMP10 are thought to signal through the Type I receptors ALK1and ALK2 and the Type II receptors ActRIIA, ActRIIB and BMPRII.

As demonstrated herein, a BMP9 polypeptide (and, as inferred by homologyand common signaling pathway, a BMP10 polypeptide) is effective atincreasing red blood cell levels in vivo and is expected to havebeneficial effects in a variety of models for anemias. It should benoted that hematopoiesis is a complex process, regulated by a variety offactors, including erythropoietin, G-CSF and iron homeostasis. The terms“increase red blood cell levels” and “promote red blood cell formation”refer to clinically observable metrics, such as hematocrit, red bloodcell counts and hemoglobin measurements, and are intended to be neutralas to the mechanism by which such changes occur.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. BMP9 and BMP10 Polypeptides and Nucleic Acids

In certain aspects, the invention relates to BMP9 polypeptides and BMP10polypeptides, including, for example, mature human BMP9 and BMP10proteins as well as BMP9 or BMP10 polypeptides that retain theprodomain, whether covalently or non-covalently attached, and variantsand truncations of the foregoing. Such variations and truncations may beselected to retain the ability to stimulate signaling by one or more ofthe known receptors for BMP9 or BMP10, including ALK1, ALK2, ActRIIA,BMPR2 and ActRIIB Optionally, a BMP9 or BMP10 polypeptide can increaseexpression of luciferase in a cell line transfected with aBRE-luciferase reporter gene construct.

As used herein, the terms “BMP-9” or “BMP-10” refer to the family ofBMP-9 or BMP-10 proteins, respectively, from any species and variantsderived from such proteins by mutagenesis, truncation or othermodification. BMP-9 proteins and BMP-10 proteins are well-conservedacross vertebrate lineages, particularly in the mature portion of theprotein, as shown in FIGS. 1 and 2. The mature portions of human BMP-9and BMP-10 also show substantial identity to each other (64%) (FIG. 3).Members of the BMP-9 or BMP-10 families are generally secreted proteins,composed of a signal peptide, a pro-domain that binds to the matureportion in a manner that competes with binding to type II receptors(e.g., BMPR2, ActRIIA, ActRIIB) and a mature portion containing acysteine knot. The mature portion binds to both a type I receptor (e.g.,ALK1 or ALK2) and a type II receptor (e.g., BMPR2, ActRIIA or ActRIIB)to form a signaling complex.

The term “BMP9 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of a BMP-9 family member, respectively,as well as any variants thereof (including mutants, fragments, fusions,and peptidomimetic forms) that retain a useful activity. For example,BMP9 polypeptides may comprise polypeptides derived from the sequence ofany known BMP9 protein and may include forms expressed with a signalpeptide, as a proprotein form (containing both the prodomain and themature portion) and as the fully mature form. As shown in FIG. 1,vertebrates as diverse as humans, mice and chickens have highlyconserved BMP9 proteins, and therefore functional variants may, forexample, be selected by reference to amino acids that are less conservedamong different vertebrate species as such changes will generally betolerated. BMP9 polypeptides may comprise, consist essentially of, orconsist of, an amino acid sequence that is at least 63%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of anaturally occurring BMP9 polypeptide such as any of SEQ. ID. Nos. 1, 2,3, 7 or 8 or the mature portions of SEQ. ID. Nos. 7 or 8. Numbering ofamino acids for all human BMP9 polypeptides described herein is based onthe numbering for SEQ ID NO:1, unless specifically designated otherwise.

Examples of BMP9 polypeptides include:

Full-length human BMP9 precursor (including signal sequence,corresponding to amino acids 1-22) (Genbank NP_057288):

(SEQ. ID. NO: 1)   1MCPGALWVAL PLLSLLAGSL QGKPLQSWGR GSAGGNAHSP LGVPGGGLPE HTFNLKMFLE  61NVKVDFLRSL NLSGVPSQDK TRVEPPQYMI DLYNRYTSDK STTPASNIVR SFSMEDAISI 121TATEDFPFQK HILLFNISIP RHEQITRAEL RLYVSCQNHV DPSHDLKGSV VIYDVLDGTD 181AWDSATETKT FLVSQDIQDE GWETLEVSSA VKRWVRSDST KSKNKLEVTV ESHRKGCDTL 241DISVPPGSRN LPFFVVESND HSSGTKETRL ELREMISHEQ ESVLKKLSKD GSTEAGESSH 301EEDTDGHVAA GSTLARRKRS AGAGSHCQKT SLRVNFEDIG WDSWIIAPKE YEAYECKGGC 361FFPLADDVTP TKHAIVQTLV HLKFPTKVGK ACCVPTKLSP ISVLYKDDMG VPTLKYHYEG 421MSVAECGCRFull-length human BMP9 proprotein (signal sequence removed but includingpro-domain, corresponding to amino acids 23-429 of SEQ ID NO:1):

(SEQ. ID. NO: 2) KPLQSWGRGS AGGNAHSPLG VPGGGLPEHTFNLKMFLENV KVDFLRSLNL SGVPSQDKTR VEPPQYMIDL YNRYTSDKST TPASNIVRSFSMEDAISITA TEDFPFQKHI LLFNISIPRH EQITRAELRL YVSCQNHVDP SHDLKGSVVIYDVLDGTDAW DSATETKTFL VSQDIQDEGW ETLEVSSAVK RWVRSDSTKS KNKLEVTVESHRKGCDTLDI SVPPGSRNLP FFVVFSNDHS SGTKETRLEL REMISHEQES VLKKLSKDGSTEAGESSHEE DTDGHVAAGS TLARRKRSAG AGSHCQKTSL RVNFEDIGWD SWIIAPKEYEAYECKGGCFF PLADDVTPTK HAIVQTLVHL KFPTKVGKAC CVPTKLSPIS VLYKDDMGVPTLKYHYEGMS VAECGCRMature human BMP9 (both signal sequence and pro-domain removed,corresponding to amino acids 320-429 of SEQ ID NO:1):

(SEQ. ID. NO: 3) SAGAGSHCQK TSLRVNFEDI GWDSWIIAPK EYEAYECKGGCFFPLADDVT PTKHAIVQTL VHLKFPTKVG KACCVPTKLSPISVLYKDDM GVPTLKYHYE GMSVAECGCR

The term “BMP10 polypeptide” includes polypeptides comprising anynaturally occurring polypeptide of a BMP10 family member, respectively,as well as any variants thereof (including mutants, fragments, fusions,and peptidomimetic forms) that retain a useful activity. For example,BMP10 polypeptides may comprise polypeptides derived from the sequenceof any known BMP10 protein and may include forms expressed with a signalpeptide, as a proprotein form and as the fully mature form. As shown inFIG. 2, vertebrates as diverse as humans, mice and chickens have highlyconserved BMP10 proteins, and therefore functional variants may, forexample, be selected by reference to amino acids that are less conservedamong different vertebrate species. BMP10 polypeptides may comprise,consist essentially of, or consist of, an amino acid sequence that is atleast 63%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%identical to the sequence of a naturally occurring BMP10 polypeptidesuch as any of SEQ. ID. Nos. 4, 5, 6, 9 or 10 or the mature portions ofSEQ. ID. Nos. 9 or 10. Numbering of amino acids for all human BMP10polypeptides described herein is based on the numbering for SEQ ID NO:4,unless specifically designated otherwise.

Examples of BMP10 polypeptides include:

Full-length human BMP10 precursor (including signal sequence,corresponding to amino acids 1-21) (Genbank NP_055297):

(SEQ. ID. NO: 4)   1MGSLVLTLCA LFCLAAYLVS GSPIMNLEQS PLEEDMSLFG DVFSEQDGVD FNTLLQSMKD  61EFLKTLNLSD IPTQDSAKVD PPEYMLELYN KFATDRTSMP SANIIRSFKN EDLFSQPVSF 121NGLRKYPLLF NVSIPHHEEV IMAELRLYTL VQRDRMIYDG VDRKITIFEV LESKGDNEGE 181RNMLVLVSGE IYGINSEWET FDVTDAIRRW QKSGSSTHQL EVHIESKHDE AEDASSGRLE 241IDTSAQNKHN PLLIVFSDDQ SSDKERKEEL NEMISHEQLP ELDNLGLDSF SSGPGEEALL 301QMRSNIIYDS TARIRRNAKG NYCKRTPLYI DFKEIGWDSW IIAPPGYEAY ECRGVCNYPL 361AEHLTPTKHA IIQALVHLKN SQKASKACCV PTKLEPISIL YLDKGVVTYK FKYEGMAVSE 421CGCR

Full-length human BMP10 proprotein (signal sequence removed butincluding pro-domain, corresponding to amino acids 22-424 of SEQ IDNO:4):

(SEQ. ID. NO: 5) SPIMNLEQSP LEEDMSLFGD VFSEQDGVDFNTLLQSMKDE FLKTLNLSDI PTQDSAKVDP PEYMLELYNK FATDRTSMPS ANIIRSFKNEDLFSQPVSFN GLRKYPLLFN VSIPHHEEVI MAELRLYTLV QRDRMIYDGV DRKITIFEVLESKGDNEGER NMLVLVSGEI YGTNSEWETF DVTDAIRRWQ KSGSSTHQLE VHIESKHDEAEDASSGRLEI DTSAQNKHNP LLIVFSDDQS SDKERKEELN EMISHEQLPE LDNLGLDSFSSGPGEEALLQ MRSNIIYDST ARIRRNAKGN YCKRTPLYID FKEIGWDSWI IAPPGYEAYECRGVCNYPLA EHLTPTKHAI IQALVHLKNS QKASKACCVP TKLEPISILY LDKGVVTYKFKYEGMAVSEC GCR

Mature human BMP10 (both signal sequence and pro-domain removed,corresponding to amino acids 317-424 of SEQ ID NO:4):

(SEQ ID NO: 6) NAKGNYCKRT PLYIDFKEIG WDSWIIAPPG YEAYECRGVCNYPLAEHLTP TKHAIIQALV HLKNSQKASK ACCVPTKLEPISILYLDKGV VTYKFKYEGM AVSECGCR

In certain aspects, the disclosure provides isolated and/or recombinantnucleic acids encoding any of the BMP9 or BMP10 polypeptides disclosedherein. Such nucleic acids may be DNA or RNA molecules. These nucleicacids may be used, for example, in methods for making BMP9 or BMP10polypeptides or as direct therapeutic agents (e.g., in a gene therapyapproach).

A nucleic acid sequence encoding a human BMP9 precursor protein is asfollows: (Genbank NM_016204)

(SEQ. ID. NO: 11)    1cggtccagcc cggcagcggg tgagagtggg tgctggccag gacggttcct tcagagcaaa   61cagcagggag atgccggccc gctccttccc agctcctccc cgtgcccgct aacacagcac  121ggccgcctgc agtctcctct ctgggtgatt gcgcgggcct aagatgtgtc ctggggcact  181gtgggtggcc ctgcccctgc tgtccctgct ggctggctcc ctacagggga agccactgca  241gagctgggga cgagggtctg ctgggggaaa cgcccacagc ccactggggg tgcctggagg  301tgggctgcct gagcacacct tcaacctgaa gatgtttctg gagaacgtga aggtggattt  361cctgcgcagc cttaacctga gtggggtccc ttcgcaggac aaaaccaggg tggagccgcc  421gcagtacatg attgacctgt acaacaggta cacgtccgat aagtcgacta cgccagcgtc  481caacattgtg cggagcttca gcatggaaga tgccatctcc ataactgcca cagaggactt  541ccccttccag aagcacatct tgctcttcaa catctccatt cctaggcatg agcagatcac  601cagagctgag ctccgactct atgtctcctg tcaaaatcac gtggacccct ctcatgacct  661gaaaggaagc gtggtcattt atgatgttct ggatggaaca gatgcctggg atagtgctac  721agagaccaag accttcctgg tgtcccagga cattcaggat gagggctggg agaccttgga  781agtgtccagc gccgtgaagc gctgggtccg gtccgactcc accaagagca aaaataagct  841ggaagtgact gtggagagcc acaggaaggg ctgcgacacg ctggacatca gtgtcccccc  901aggttccaga aacctgccct tctttgttgt cttctccaat gaccacagca gtgggaccaa  961ggagaccagg ctggagctga gggagatgat cagccatgaa caagagagcg tgctcaagaa 1021gctgtccaag gacggctcca cagaggcagg tgagagcagt cacgaggagg acacggatgg 1081ccacgtggct gcggggtcga ctttagccag gcggaaaagg agcgccgggg ctggcagcca 1141ctgtcaaaag acctccctgc gggtaaactt cgaggacatc ggctgggaca gctggatcat 1201tgcacccaag gagtatgaag cctacgagtg taagggcggc tgcttcttcc ccttggctga 1261cgatgtgacg ccgacgaaac acgctatcgt gcagaccctg gtgcatctca agttccccac 1321aaaggtgggc aaggcctgct gtgtgcccac caaactgagc cccatctccg tcctctacaa 1381ggatgacatg ggggtgccca ccctcaagta ccattacgag ggcatgagcg tggcagagtg 1441tgggtgcagg tag

The coding region for BMP9 (signal peptide, prodomain and matureportion) runs from position 164-1453 of SEQ. ID. No. 11. The signalpeptide is encoded by nucleotides 164-229, the prodomain by nucleotides230-1120 and the mature peptide by positions 1121-1450.

The nucleic acid sequence encoding a human BMP10 precursor protein is asfollows (Genbank NM_014482):

(SEQ. ID. NO: 12)    1ggggagagga agagtggtag ggggagggag agagagagga agagtttcca aacttgtctc   61cagtgacagg agacatttac gttccacaag ataaaactgc cacttagagc ccagggaagc  121taaaccttcc tggcttggcc taggagctcg agcggagtca tgggctctct ggtcctgaca  181ctgtgcgctc ttttctgcct ggcagcttac ttggtttctg gcagccccat catgaaccta  241gagcagtctc ctctggaaga agatatgtcc ctctttggtg atgttttctc agagcaagac  301ggtgtcgact ttaacacact gctccagagc atgaaggatg agtttcttaa gacactaaac  361ctctctgaca tccccacgca ggattcagcc aaggtggacc caccagagta catgttggaa  421ctctacaaca aatttgcaac agatcggacc tccatgccct ctgccaacat cattaggagt  481ttcaagaatg aagatctgtt ttcccagccg gtcagtttta atgggctccg aaaatacccc  541ctcctcttca atgtgtccat tcctcaccat gaagaggtca tcatggctga acttaggcta  601tacacactgg tgcaaaggga tcgtatgata tacgatggag tagaccggaa aattaccatt  661tttgaagtgc tggagagcaa aggggataat gagggagaaa gaaacatgct ggtcttggtg  721tctggggaga tatatggaac caacagtgag tgggagactt ttgatgtcac agatgccatc  781agacgttggc aaaagtcagg ctcatccacc caccagctgg aggtccacat tgagagcaaa  841cacgatgaag ctgaggatgc cagcagtgga cggctagaaa tagataccag tgcccagaat  901aagcataacc ctttgctcat cgtgttttct gatgaccaaa gcagtgacaa ggagaggaag  961gaggaactga atgaaatgat ttcccatgag caacttccag agctggacaa cttgggcctg 1021gatagctttt ccagtggacc tggggaagag gctttgttgc agatgagatc aaacatcatc 1081tatgactcca ctgcccgaat cagaaggaac gccaaaggaa actactgtaa gaggaccccg 1141ctctacatcg acttcaagga gattgggtgg gactcctgga tcatcgctcc gcctggatac 1201gaagcctatg aatgccgtgg tgtttgtaac taccccctgg cagagcatct cacacccaca 1261aagcatgcaa ttatccaggc cttggtccac ctcaagaatt cccagaaagc ttccaaagcc 1321tgctgtgtgc ccacaaagct agagcccatc tccatcctct atttagacaa aggcgtcgtc 1381acctacaagt ttaaatacga aggcatggcc gtctccgaat gtggctgtag atagaagaag 1441agtcctatgg cttatttaat aactgtaaat gtgtatattt ggtgttccta tttaatgaga 1501ttatttaata agggtgtaca gtaatagagg cttgctgcct tcaggaaatg gacaggtcag 1561tttgttgtag gaaatgcata tttt

The coding region for BMP10 (signal peptide, prodomain and matureportion) runs from position 160-1434 of SEQ. ID. No. 12. The signalpeptide is encoded by nucleotides 160-222, the prodomain by nucleotides223-1107 and the mature peptide by positions 1108-1431.

In certain aspects, the subject nucleic acids encoding BMP9 or BMP10polypeptides are further understood to include nucleic acids that arevariants of SEQ ID NOs: 11 or 12. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants; and will, therefore, includecoding sequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NOs: 11 or 12.

In certain embodiments, the disclosure provides isolated or recombinantnucleic acid sequences that are at least 63%, 70%. 80%, 85%, 90%, 95%,97%, 98%, 99% or 100% identical to SEQ ID NO: 11 or 12 or the portionsthereof that encode the prodomain or mature portion. One of ordinaryskill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NO: 11 or 12, and variants of SEQ ID NO: 11 or12, are also within the scope of this invention. In further embodiments,the nucleic acid sequences of the invention can be isolated,recombinant, and/or fused with a heterologous nucleotide sequence, or ina DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under stringent conditions to thenucleotide sequence designated in SEQ ID NO: 11 or 12, including theportions thereof that encode the prodomain or mature portion, complementsequence of SEQ ID NO: 11 or 12, including the portions thereof thatencode the prodomain or mature portion thereof. In a particularembodiment, the disclosure provides nucleic acids that hybridize understringent conditions to a complement to the nucleic acid of 1121-1450 ofSEQ ID NO:11 or a complement of the nucleic acid of 1108-1431 of SEQ IDNO:12, and BMP9 or BMP10 polypeptides encoded by the foregoing. Asdiscussed above, one of ordinary skill in the art will understandreadily that appropriate stringency conditions which promote DNAhybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the disclosureprovides nucleic acids which hybridize under low stringency conditionsof 6×SSC at room temperature followed by a wash at 2×SSC at roomtemperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 11 or 12 due to degeneracy in the genetic code are alsowithin the scope of the invention. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. In certain embodiments, the BMP9 orBMP10 polypeptide will be encoded by an alternative nucleotide sequence.Alternative nucleotide sequences are degenerate with respect to thenative BMP9 or BMP10 nucleic acid sequence but still encode for the samefusion protein.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the disclosure, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding a BMP9 or BMP10 polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the BMP9 or BMP10 polypeptide.Accordingly, the term regulatory sequence includes promoters, enhancers,and other expression control elements. Exemplary regulatory sequencesare described in Goeddel; Gene Expression Technology: Methods inEnzymology, Academic Press, San Diego, Calif. (1990). For instance, anyof a wide variety of expression control sequences that control theexpression of a DNA sequence when operatively linked to it may be usedin these vectors to express DNA sequences encoding a BMP9 or BMP10polypeptide. Such useful expression control sequences, include, forexample, the early and late promoters of SV40, tet promoter, adenovirusor cytomegalovirus immediate early promoter, RSV promoters, the lacsystem, the trp system, the TAC or TRC system, T7 promoter whoseexpression is directed by T7 RNA polymerase, the major operator andpromoter regions of phage lambda, the control regions for fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast α-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid for production of BMP9 or BMP10 polypeptidescan be produced by ligating the cloned gene, or a portion thereof, intoa vector suitable for expression in either prokaryotic cells, eukaryoticcells (yeast, avian, insect or mammalian), or both. Expression vehiclesfor production of a recombinant BMP9 or BMP10 polypeptide includeplasmids and other vectors. For instance, suitable vectors includeplasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids,pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmidsfor expression in prokaryotic cells, such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject BMP9 or BMP10 polypeptides in CHO cells, such as aPcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors(Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison,Wis.). As will be apparent, the subject gene constructs can be used tocause expression of the subject BMP9 or BMP10 polypeptides in cellspropagated in culture, e.g., to produce proteins, including fusionproteins or variant proteins, for purification.

This disclosure also pertains to a host cell transfected with arecombinant gene including a coding sequence for one or more of thesubject BMP9 or BMP10 polypeptides. The host cell may be any prokaryoticor eukaryotic cell. For example, a BMP9 or BMP10 polypeptide of theinvention may be expressed in bacterial cells such as E. coli, insectcells (e.g., using a baculovirus expression system), yeast, or mammaliancells. Other suitable host cells are known to those skilled in the art.

The above-described nucleic acids may be used to express BMP9 or BMP10polypeptides in suitable cells, including, for example, HEK cells, COScells and CHO cells. The signal sequence can be a native signal sequenceof BMP9 or BMP10, or a signal sequence from another protein, such as atissue plasminogen activator (TPA) signal sequence or a honey beemelittin (HBM) signal sequence. The prodomain sequences of BMP9 andBMP10 may be interchanged, such that a BMP10 mature portion is expressedwith a BMP9 prodomain or vice versa. The protein PACE (or Furin)mediates cleavage of the proprotein into two peptides, the proproteinand the mature portion, and thus it is useful to express a PACEtransgene in a cell that is intended to produce a BMP9 or BMP10polypeptide if such cleavage is desired. It is generally accepted thatmembers of the GDF or BMP families need to dissociate from theirprodomains in order to become fully active. In the case of BMP9 orBMP10, the prodomain remains associated with the mature portion, thus itmay be desirable to separate the mature portion to generate theadministrable pharmaceutical form. Alternatively, it is recognized herethat the prodomain may confer desirable pharmaceutical properties,including, for example, longer serum half-life and greaterbioavailability, and thus in certain embodiments the disclosure providespharmaceutical preparations comprising the mature portion of a BMP9 orBMP10 polypeptide that is covalently or non-covalently associated with aprodomain polypeptide. A “prodomain polypeptide” is a polypeptidecomprising, consisting essentially of, or consisting of, an amino acidsequence that is at least 63%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, or 100% identical to the sequence of a naturally occurring BMP9 orBMP10 prodomain such as amino acids 23-319 of SEQ ID No. 1 or aminoacids 22-316 of SEQ ID No. 4. It will be apparent that a prodomainpolypeptide should not generally include more than 30, 20, 10 or 5 aminoacids of the corresponding mature portion. In certain embodiments, aprodomain polypeptide will bind to the mature portion of a BMP9 or BMP10polypeptide with a KD of no greater than 10⁻⁶M, 10⁻⁷M, 10⁻⁸M or 10⁻⁹M,or less.

In certain embodiments, the present disclosure contemplates makingfunctional variants by modifying the structure of a BMP9 or BMP10polypeptide for such purposes as enhancing therapeutic efficacy, orstability (e.g., ex vivo shelf life and resistance to proteolyticdegradation in vivo). BMP9 or BMP10 polypeptides can also be generatedby amino acid substitution, deletion, or addition. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of a BMP9 or BMP10 polypeptide results in afunctional variant can be readily determined by assessing the ability ofthe BMP9 or BMP10 polypeptide to produce a response in cells relative tothe unmodified BMP9 or BMP10 polypeptide, or to bind to one or morereceptors. In the case of variations in a prodomain polypeptide, thefunctional activity of a variant may be assessed by measuring theability of the prodomain to bind to a mature BMP9 or BMP10 polypeptide.

In certain embodiments, the present invention contemplates BMP9 or BMP10polypeptides having specific mutations so as to alter the glycosylationof the BMP9 or BMP10 polypeptide. Alterations in amino acid sequence maybe made so as to introduce one or more N-linked glycosylation sites,which are generally an NXS or NXT sequence. Mutations may also beselected so as to eliminate one or more glycosylation sites, such asO-linked or N-linked glycosylation sites. The alteration may also bemade by the addition of, or substitution by, one or more asparagine,serine or threonine residues to the sequence of a BMP9 or BMP10polypeptide. A variety of amino acid substitutions or deletions at oneor both of the first or third amino acid positions of a glycosylationrecognition site (and/or amino acid deletion at the second position)results in non-glycosylation at the modified tripeptide sequence.Another means of increasing the number of carbohydrate moieties on aBMP9 or BMP10 polypeptide is by chemical or enzymatic coupling ofglycosides to the BMP9 or BMP10 polypeptide. Depending on the couplingmode used, the sugar(s) may be attached to (a) arginine and histidine;(b) free carboxyl groups; (c) free sulfhydryl groups such as those ofcysteine; (d) free hydroxyl groups such as those of serine, threonine,or hydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 and in Aplin and Wriston (1981) CRCCrit. Rev. Biochem., pp. 259-306, incorporated by reference herein.Removal of one or more carbohydrate moieties present on a BMP9 or BMP10polypeptide may be accomplished chemically and/or enzymatically.Chemical deglycosylation may involve, for example, exposure of the BMP9or BMP10 polypeptide to the compound trifluoromethanesulfonic acid, oran equivalent compound. This treatment results in the cleavage of mostor all sugars except the linking sugar (N-acetylglucosamine orN-acetylgalactosamine), while leaving the amino acid sequence intact.Chemical deglycosylation is further described by Hakimuddin et al.(1987) Arch. Biochem. Biophys. 259:52 and by Edge et al. (1981) Anal.Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on BMP9 orBMP10 polypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. (1987) Meth. Enzymol.138:350. The sequence of a BMP9 or BMP10 polypeptide may be adjusted, asappropriate, depending on the type of expression system used, asmammalian, yeast, insect and plant cells may all introduce differingglycosylation patterns that can be affected by the amino acid sequenceof the peptide. In general, BMP9 or BMP10 polypeptides for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of a BMP9 or BMP10polypeptide, including, optionally, truncation variants; pools ofcombinatorial mutants are especially useful for identifying BMP9 orBMP10 sequences. The purpose of screening such combinatorial librariesmay be to generate, for example, BMP9 or BMP10 polypeptide variantswhich have altered properties, such as altered pharmacokinetics, oraltered receptor binding. A variety of screening assays are providedbelow, and such assays may be used to evaluate variants. For example, aBMP9 or BMP10 polypeptide variant may be screened for the ability tobind to an ALK1, ActRIIA or ActRIIB polypeptide.

The activity of a BMP9 or BMP10 polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of aBMP9 or BMP10 polypeptide variant on the expression of genes involved inhematopoiesis may be assessed. Likewise, a BMP9 or BMP10 polypeptide maybe administered to a mouse or other animal, and one or more bloodmeasurements, such as an RBC count, hemoglobin levels, hematocritlevels, iron stores, or reticulocyte count may be assessed using artrecognized methods. The BMP-responsive element (BRE) element, generallyobtained from the promoter region of the ID1 gene is widely recognizedas an appropriate reporter gene for members of the BMP/GDF family thatstimulate SMAD 1/5/8 signaling. See, e.g., Logeart-Avramoglou D, et al.,Anal Biochem. 2006 Feb. 1; 349(1):78-86. BMP9 or BMP10 polypeptide mayalso be measured by induction of alkaline phosphatase by ATDC5 mousechondrogenic cells or MC3T3-E1 mouse osteoblastic cells. Nakamura, K. etal. (1999) Exp. Cell Res. 250:351.

In certain embodiments, the BMP9 or BMP10 polypeptides may furthercomprise post-translational modifications in addition to any that arenaturally present in the BMP9 or BMP10 polypeptides. Such modificationsinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, acylation and modificationwith polyethylene glycol (PEG). As a result, BMP9 or BMP10 polypeptidesmay contain non-amino acid elements, such as polyethylene glycols,lipids, poly- or mono-saccharide, and phosphates. Effects of suchnon-amino acid elements on the functionality of a BMP9 orBMP10polypeptide may be tested as described herein for other BMP9 orBMP10 polypeptide variants. When a BMP9 or BMP10 polypeptide is producedin cells by cleaving a nascent form of BMP9 or BMP10 polypeptide,post-translational processing may also be important for correct foldingand/or function of the protein. Different cells (such as CHO, HeLa,MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and maybe chosen to ensure the correct modification and processing of the BMP9or BMP10 polypeptides.

In certain aspects, BMP9 or BMP10 polypeptides include fusion proteinshaving at least a portion of a BMP9 or BMP10 polypeptide and one or morefusion domains. Well known examples of such fusion domains include, butare not limited to, polyhistidine, Glu-Glu, glutathione S transferase(GST), thioredoxin, protein A, protein G, an immunoglobulin heavy chainconstant region (e.g., an Fc), maltose binding protein (MBP), or humanserum albumin. A fusion domain may be selected so as to confer a desiredproperty. For example, some fusion domains are particularly useful forisolation of the fusion proteins by affinity chromatography. For thepurpose of affinity purification, relevant matrices for affinitychromatography, such as glutathione-, amylase-, and nickel- orcobalt-conjugated resins are used. Many of such matrices are availablein “kit” form, such as the Pharmacia GST purification system and theQIAexpress™ system (Qiagen) useful with (HIS₆) fusion partners. Asanother example, a fusion domain may be selected so as to facilitatedetection of the BMP9 or BMP10 polypeptides. Examples of such detectiondomains include the various fluorescent proteins (e.g., GFP) as well as“epitope tags,” which are usually short peptide sequences for which aspecific antibody is available. Well known epitope tags for whichspecific monoclonal antibodies are readily available include FLAG,influenza virus haemagglutinin (HA), and c-myc tags. In some cases, thefusion domains have a protease cleavage site, such as for Factor Xa orThrombin, which allows the relevant protease to partially digest thefusion proteins and thereby liberate the recombinant proteins therefrom.The liberated proteins can then be isolated from the fusion domain bysubsequent chromatographic separation. In certain preferred embodiments,a BMP9 or BMP10 polypeptide is fused with a domain that stabilizes theBMP9 or BMP10 polypeptide in vivo (a “stabilizer” domain). By“stabilizing” is meant anything that increases serum half life,regardless of whether this is because of decreased destruction,decreased clearance by the kidney, or other pharmacokinetic effect.Fusions with the Fc portion of an immunoglobulin are known to conferdesirable pharmacokinetic properties on a wide range of proteins.Likewise, fusions to human serum albumin can confer desirableproperties. Other types of fusion domains that may be selected includemultimerizing (e.g., dimerizing, tetramerizing) domains and functionaldomains (that confer an additional biological function, such as furtherincreasing red blood cell levels).

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, a BMP9 or BMP10 polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to a BMP9 or BMP10 polypeptide. The BMP9or BMP10 polypeptide domain and the heterologous domain need not beadjacent in a fusion protein, and additional domains or amino acidsequences may be included C- or N-terminal to either domain or betweenthe domains.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the BMP9 or BMP10 polypeptides, which areisolated from, or otherwise substantially free of, other proteins.

In certain embodiments, BMP9 or BMP10 polypeptides (unmodified ormodified) of the invention can be produced by a variety of art-knowntechniques. For example, polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the BMP9 or BMP10 polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary (CHO)cells, COS cells, baculovirus) as is well known in the art, followed byprotein purification. BMP9 and BMP10 are also commercially availablefrom R&D Systems (Minneapolis, Minn.).

Accordingly, the disclosure provides methods of producing the subjectBMP9 or BMP10 polypeptides. For example, a host cell transfected with anexpression vector encoding a BMP9 or BMP10 polypeptide can be culturedunder appropriate conditions to allow expression of the polypeptide tooccur. The BMP9 or BMP10 polypeptide may be secreted and isolated from amixture of cells and medium containing the BMP9 or BMP10 polypeptide.Alternatively, the polypeptide may be retained cytoplasmically or in amembrane fraction and the cells harvested, lysed and the proteinisolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The subject BMP9 or BMP10 polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the BMP9 or BMP10 polypeptides.

The disclosure further provides novel methods for purification of BMP9or BMP10 polypeptides by using the affinity of these proteins for one ormore of their receptors, including ALK1, ALK2, BMPR2, ActRIIA or ActRIIBA solid matrix (e.g., chromatography resin) may be joined to aligand-binding portion of any of the foregoing to create an affinitymatrix that will bind selectively to BMP9 or BMP10 polypeptides. Theextracellular domain of the receptor may be fused to an Fc portion of animmunoglobulin and joined to a matrix containing an Fc binding protein,such as protein A. Surprisingly, a variant of an ActRIIB extracellulardomain which contains an aspartic acid or glutamic acid rather than aleucine at position 79 is a particularly effective reagent for affinitypurification of BMP9 or BMP10 polypeptides. Notably, this variant hasreduced affinity for BMP9 or BMP10 relative to wild-type ActRIIB See thefollowing published PCT patent applications for examples of receptorsand receptor-Fc fusion constructs that are useful in the production ofBMP9 or BMP10 polypeptides: WO 2011/020045, WO 2010/151426, WO2010/019261, WO 2009/139891, WO 2009/134428, WO 2008/097541, WO2008/076437, WO 2007/062188 and WO 2006/012627, the receptor andreceptor-Fc sequences of which are incorporated by reference. ActRIIA,BMPR2 and ActRIIB reagents are useful for purifying BMP9 or BMP10 matureproteins, as these proteins will compete with the propeptide for bindingto the mature portion. ALK1 or ALK2 reagents are useful for purifyingBMP9 or BMP10 polypeptides as complexes with the prodomain, as thesebind at a site that is distinct and non-competitive relative to thepropeptide.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant BMP9 or BMP10polypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni′ metal resin. The purification leadersequence can then be subsequently removed by treatment with enterokinaseto provide the purified BMP9 or BMP10 polypeptide (e.g., see Hochuli etal., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS USA88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

3. Exemplary Therapeutic Uses

In certain embodiments, the BMP9 or BMP10 polypeptides of the presentdisclosure can be used to increase red blood cell levels in mammals suchas rodents and primates, and particularly human patients. Additionally,BMP9 or BMP10 polypeptides may be used in combination with EPO receptoractivators to achieve an increase in red blood cells at lower doseranges or to achieve an overall higher level of RBCs or a greaterresponse rate. This may be beneficial in reducing the known off-targeteffects and risks associated with high doses of EPO receptor activators.In certain embodiments, the present invention provides methods oftreating or preventing anemia in an individual in need thereof byadministering to the individual a therapeutically effective amount of aBMP9 or BMP10 polypeptide or a combination (or concomitant therapy) of aBMP9 or BMP10 polypeptide and a EPO receptor activator. These methodsmay be used for therapeutic and prophylactic treatments of mammals, andparticularly humans.

The BMP9 or BMP10 polypeptides may be used in combination with EPOreceptor activators to reduce the required dose of these activators inpatients that are susceptible to adverse effects of EPO. The primaryadverse effects of EPO are an excessive increase in the hematocrit orhemoglobin levels and polycythemia. Elevated hematocrit levels can leadto hypertension (more particularly aggravation of hypertension) andvascular thrombosis. Other adverse effects of EPO which have beenreported, some of which related to hypertension, are headaches,influenza-like syndrome, obstruction of shunts, myocardial infarctionsand cerebral convulsions due to thrombosis, hypertensive encephalopathy,and red cell blood cell applasia (Singibarti, (1994) J. Clin Investig72(suppl 6), S36-S43; Horl et al. (2000) Nephrol Dial Transplant15(suppl 4), 51-56; Delanty et al. (1997) Neurology 49, 686-689; Bunn(2002) N Engl J Med 346(7), 522-523).

The rapid effect on red blood cell levels of the BMP9 or BMP10polypeptides disclosed herein indicate that these agents act by adifferent mechanism than EPO. Accordingly, these antagonists may beuseful for increasing red blood cell and hemoglobin levels in patientsthat do not respond well to EPO. For example, a BMP9 or BMP10polypeptide may be beneficial for a patient in which administration of anormal to increased (>300 IU/kg/week) dose of EPO does not result in theincrease of hemoglobin level up to the target level. Patients with aninadequate EPO response are found for all types of anemia, but highernumbers of non-responders have been observed particularly frequently inpatients with cancers and patients with end-stage renal disease. Aninadequate response to EPO can be either constitutive (i.e. observedupon the first treatment with EPO) or acquired (e.g. observed uponrepeated treatment with EPO).

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established. In eithercase, prevention or treatment may be discerned in the diagnosis providedby a physician or other health care provider and the intended result ofadministration of the therapeutic agent.

As shown herein, BMP9 or BMP10 polypeptides, optionally combined with anEPO receptor activator, may be used to increase red blood cell,hemoglobin or reticulocyte levels in healthy individuals, and such BMP9or BMP10 polypeptides may be used in selected patient populations.Examples of appropriate patient populations include those withundesirably low red blood cell or hemoglobin levels, such as patientshaving an anemia, and those that are at risk for developing undesirablylow red blood cell or hemoglobin levels, such as those patients that areabout to undergo major surgery or other procedures that may result insubstantial blood loss. In one embodiment, a patient with adequate redblood cell levels is treated with a BMP9 or BMP10 polypeptide toincrease red blood cell levels, and then blood is drawn and stored forlater use in transfusions.

BMP9 or BMP10 polypeptides, optionally combined with an EPO receptoractivator, disclosed herein may be used to increase red blood celllevels in patients having an anemia. When observing hemoglobin levels inhumans, a level of less than normal for the appropriate age and gendercategory may be indicative of anemia, although individual variations aretaken into account. For example, a hemoglobin level of 12 g/dl isgenerally considered the lower limit of normal in the general adultpopulation. Potential causes include blood-loss, nutritional deficits,medication reaction, various problems with the bone marrow and manydiseases. More particularly, anemia has been associated with a varietyof disorders that include, for example, chronic renal failure,myelodysplastic syndrome, rheumatoid arthritis, bone marrowtransplantation. Anemia may also be associated with the followingconditions: solid tumors (e.g. breast cancer, lung cancer, coloncancer); tumors of the lymphatic system (e.g. chronic lymphocyteleukemia, non-Hodgkins and Hodgkins lymphomas); tumors of thehematopoietic system (e.g. leukemia, myelodysplastic syndrome, multiplemyeloma); radiation therapy; chemotherapy (e.g. platinum containingregimens); inflammatory and autoimmune diseases, including, but notlimited to, rheumatoid arthritis, other inflammatory arthritides,systemic lupus erythematosis (SLE), acute or chronic skin diseases (e.g.psoriasis), inflammatory bowel disease (e.g. Crohn's disease andulcerative colitis); acute or chronic renal disease or failure includingidiopathic or congenital conditions; acute or chronic liver disease;acute or chronic bleeding; situations where transfusion of red bloodcells is not possible due to patient allo- or auto-antibodies and/or forreligious reasons (e.g. some Jehovah's Witnesses); infections (e.g.malaria, osteomyelitis); hemoglobinopathies, including, for example,sickle cell disease, thalassemias; drug use or abuse, e.g. alcoholmisuse; pediatric patients with anemia from any cause to avoidtransfusion; and elderly patients or patients with underlyingcardiopulmonary disease with anemia who cannot receive transfusions dueto concerns about circulatory overload.

BMP9 or BMP10 polypeptides, optionally combined with an EPO receptoractivator, would be appropriate for treating anemias ofhypoproliferative bone marrrow, which are typically associated withlittle change in red blood cell (RBC) morphology. Hypoproliferativeanemias include: 1) anemia of chronic disease, 2) anemia of kidneydisease, and 3) anemia associated with hypometabolic states. In each ofthese types, endogenous erythropoietin levels are inappropriately lowfor the degree of anemia observed. Other hypoproliferative anemiasinclude: 4) early-stage iron-deficient anemia, and 5) anemia caused bydamage to the bone marrow. In these types, endogenous erythropoietinlevels are appropriately elevated for the degree of anemia observed.

The most common type of anemia is anemia of chronic disease, whichencompasses inflammation, infection, tissue injury, and conditions suchas cancer, and is distinguished by both low erythropoietin levels and aninadequate response to erythropoietin in the bone marrow (Adamson, 2008,Harrison's Principles of Internal Medicine, 17th ed.; McGraw Hill, NewYork, pp 628-634). Many factors can contribute to cancer-related anemia.Some are associated with the disease process itself and the generationof inflamatory cytokines such as interleukin-1, interferon-gamma, andtumor necrosis factor (Bron et al., 2001, Semin Oncol 28(Suppl 8):1-6).Among its effects, inflammation induces the key iron-regulatory peptidehepcidin, thereby inhibiting iron export from macrophages and generallylimiting iron availability for erythropoiesis (Ganz, 2007, J Am SocNephrol 18:394-400). Blood loss through various routes can alsocontribute to cancer-related anemia. The prevalence of anemia due tocancer progression varies with cancer type, ranging from 5% in prostatecancer up to 90% in multiple myeloma. Cancer-related anemia has profoundconsequences for patients, including fatigue and reduced quality oflife, reduced treatment efficacy, and increased mortality.

Chronic kidney disease is associated with hypoproliferative anemia thatvaries in severity with the degree of renal impairment. Such anemia isprimarily due to inadequate production of erythropoietin and reducedsurvival of red blood cells. Chronic kidney disease usually proceedsgradually over a period of years or decades to end-stage (Stage-5)disease, at which point dialysis or kidney transplantation is requiredfor patient survival. Anemia often develops early in this process andworsens as disease progresses. The clinical consequences of anemia ofkidney disease are well-documented and include development of leftventricular hypertrophy, impaired cognitive function, reduced quality oflife, and altered immune function (Levin et al., 1999, Am J Kidney Dis27:347-354; Nissenson, 1992, Am J Kidney Dis 20(Suppl 1):21-24; Revickiet al., 1995, Am J Kidney Dis 25:548-554; Gafter et al., 1994, KidneyInt 45:224-231). A BMP9 or BMP10 polypeptide, optionally combined withan EPO receptor activator, can be used to treat anemia of kidneydisease.

Many conditions resulting in a hypometabolic rate can produce amild-to-moderate hypoproliferative anemia. Among such conditions areendocrine deficiency states. For example, anemia can occur in Addison'sdisease, hypothyroidism, hyperparathyroidism, or males who are castratedor treated with estrogen. Mild-to-moderate anemia can also occur withreduced dietary intake of protein, a condition particularly prevalent inthe elderly. Finally, anemia can develop in patients with chronic liverdisease arising from nearly any cause (Adamson, 2008, Harrison'sPrinciples of Internal Medicine, 17th ed.; McGraw Hill, New York, pp628-634).

Anemia resulting from acute blood loss of sufficient volume, such asfrom trauma or postpartum hemorrhage, is known as acute post-hemorrhagicanemia. Acute blood loss initially causes hypovolemia without anemiasince there is proportional depletion of RBCs along with other bloodconstituents. However, hypovolemia will rapidly trigger physiologicmechanisms that shift fluid from the extravascular to the vascularcompartment, which results in hemodilution and anemia. If chronic, bloodloss gradually depletes body iron stores and eventually leads to irondeficiency. A BMP9 or BMP10 polypeptide, optionally combined with an EPOreceptor activator, can be used to speed recovery from anemia of acuteblood loss.

Iron-deficiency anemia is the final stage in a graded progression ofincreasing iron deficiency which includes negative iron balance andiron-deficient erythropoiesis as intermediate stages. Iron deficiencycan result from increased iron demand, decreased iron intake, orincreased iron loss, as exemplified in conditions such as pregnancy,inadequate diet, intestinal malabsorption, acute or chronicinflammation, and acute or chronic blood loss. With mild-to-moderateanemia of this type, the bone marrow remains hypoproliferative, and RBCmorphology is largely normal; however, even mild anemia can result insome microcytic hypochromic RBCs, and the transition to severeiron-deficient anemia is accompanied by hyperproliferation of the bonemarrow and increasingly prevalent microcytic and hypochromic RBCs(Adamson, 2008, Harrison's Principles of Internal Medicine, 17th ed.;McGraw Hill, New York, pp 628-634). Appropriate therapy foriron-deficiency anemia depends on its cause and severity, with oral ironpreparations, parenteral iron formulations, and RBC transfusion as majorconventional options. A BMP9 or BMP10 polypeptide, optionally combinedwith an EPO receptor activator, could be used to treat chroniciron-deficiency anemias alone or in combination with conventionaltherapeutic approaches, particularly to treat anemias of multifactorialorigin.

Hypoproliferative anemias can result from primary dysfunction or failureof the bone marrow, instead of dysfunction secondary to inflammation,infection, or cancer progression. Prominent examples would bemyelosuppression caused by cancer chemotherapeutic drugs or cancerradiation therapy. A broad review of clinical trials found that mildanemia can occur in 100% of patients after chemotherapy, while moresevere anemia can occur in up to 80% of such patients (Groopman et al.,1999, J Natl Cancer Inst 91:1616-1634). Myelosuppressive drugsinclude: 1) alkylating agents such as nitrogen mustards (e.g.,melphalan) and nitrosoureas (e.g., streptozocin); 2) antimetabolitessuch as folic acid antagonists (e.g., methotrexate), purine analogs(e.g., thioguanine), and pyrimidine analogs (e.g., gemcitabine); 3)cytotoxic antibotics such as anthracyclines (e.g., doxorubicin); 4)kinase inhibitors (e.g., gefitinib); 5) mitotic inhibitors such astaxanes (e.g., paclitaxel) and vinca alkaloids (e.g., vinorelbine); 6)monoclonal antibodies (e.g., rituximab); and 7) topoisomerase inhibitors(e.g., topotecan and etoposide). A BMP9 or BMP10 polypeptide, optionallycombined with an EPO receptor activator, can be used to treat anemiacaused by chemotherapeutic agents and/or radiation therapy.

BMP9 or BMP10 polypeptides, optionally combined with an EPO receptoractivator, would also be appropriate for treating anemias of disorderedRBC maturation, which are characterized in part by undersized(microcytic), oversized (macrocytic), misshapen, or abnormally colored(hypochromic) RBCs.

Patients may be treated with a dosing regimen intended to restore thepatient to a target hemoglobin level, usually between about 10 g/dl andabout 12.5 g/dl, and typically about 11.0 g/dl (see also Jacobs et al.(2000) Nephrol Dial Transplant 15, 15-19), although lower target levelsmay cause fewer cardiovascular side effects. Alternatively, hematocritlevels (percentage of the volume of a blood sample occupied by thecells) can be used as a measure for the condition of red blood cells.Hematocrit levels for healthy individuals range from 41 to 51% for adultmales and from 35 to 45% for adult females. Target hematocrit levels areusually around 30-33%. Moreover, hemoglobin/hematocrit levels vary fromperson to person. Thus, optimally, the target hemoglobin/hematocritlevel can be individualized for each patient.

In certain embodiments, the present invention provides methods formanaging a patient that has been treated with, or is a candidate to betreated with, a BMP9 or BMP10 polypeptide by measuring one or morehematologic parameters in the patient. The hematologic parameters may beused to evaluate appropriate dosing for a patient who is a candidate tobe treated with a BMP9 or BMP10 polypeptide, to monitor the hematologicparameters during treatment with a BMP9 or BMP10 polypeptide, toevaluate whether to adjust the dosage during treatment with a BMP9 orBMP10 polypeptide, and/or to evaluate an appropriate maintenance dose ofa BMP9 or BMP10 polypeptide. If one or more of the hematologicparameters are outside the normal level, dosing with a BMP9 or BMP10polypeptide may be reduced, delayed or terminated.

Hematologic parameters that may be measured in accordance with themethods provided herein include, for example, red blood cell levels,blood pressure, iron stores, and other agents found in bodily fluidsthat correlate with increased red blood cell levels, using artrecognized methods. Such parameters may be determined using a bloodsample from a patient. Increases in red blood cell levels, hemoglobinlevels, and/or hematocrit levels may cause increases in blood pressure.

In one embodiment, if one or more hematologic parameters are outside thenormal range, or on the high side of normal, in a patient who is acandidate to be treated with a BMP9 or BMP10 polypeptide then onset ofadministration of the polypeptide may be delayed until the hematologicparameters have returned to a normal or acceptable level eithernaturally or via therapeutic intervention. For example, if a candidatepatient is hypertensive or prehypertensive, then the patient may betreated with a blood pressure lowering agent in order to reduce thepatient's blood pressure. Any blood pressure lowering agent appropriatefor the individual patient's condition may be used including, forexample, diuretics, adrenergic inhibitors (including alpha blockers andbeta blockers), vasodilators, calcium channel blockers,angiotensin-converting enzyme (ACE) inhibitors, or angiotensin IIreceptor blockers. Blood pressure may alternatively be treated using adiet and exercise regimen. Similarly, if a candidate patient has ironstores that are lower than normal, or on the low side of normal, thenthe patient may be treated with an appropriate regimen of diet and/oriron supplements until the patient's iron stores have returned to anormal or acceptable level. For patients having higher than normal redblood cell levels and/or hemoglobin levels, then administration of theBMP9 or BMP10 polypeptide may be delayed until the levels have returnedto a normal or acceptable level.

In certain embodiments, if one or more hematologic parameters areoutside the normal range, or on the high side of normal, in a patientwho is a candidate to be treated with a BMP9 or BMP10 polypeptide thenthe onset of administration may be delayed. However, the dosage amountor frequency of dosing of the BMP9 or BMP10 polypeptide may be set at anamount that would reduce the risk of an unacceptable increase in thehematologic parameters arising upon administration of the BMP9 or BMP10polypeptide. Alternatively, a therapeutic regimen may be developed forthe patient that combines a BMP9 or BMP10 polypeptide with a therapeuticagent that addresses the undesirable level of the hematologic parameter.For example, if the patient has elevated blood pressure, or the BMP9 orBMP10 polypeptide appears to be causing elevated blood pressure, then atherapeutic regimen involving administration of a BMP9 or BMP10polypeptide and a blood pressure lowering agent may be designed. For apatient having lower than desired iron stores, a therapeutic regimen ofa BMP9 or BMP10 polypeptide and iron supplementation may be developed.

In one embodiment, baseline parameter(s) for one or more hematologicparameters may be established for a patient who is a candidate to betreated with a BMP9 or BMP10 polypeptide and an appropriate dosingregimen establish for that patient based on the baseline value(s).Alternatively, established baseline parameters based on a patient'smedical history could be used to inform an appropriate BMP9 or BMP10polypeptide dosing regimen for a patient. For example, if a healthypatient has an established baseline blood pressure reading that is abovethe defined normal range it may not be necessary to bring the patient'sblood pressure into the range that is considered normal for the generalpopulation prior to treatment with the BMP9 or BMP10 polypeptide. Apatient's baseline values for one or more hematologic parameters priorto treatment with a BMP9 or BMP10 polypeptide may also be used as therelevant comparative values for monitoring any changes to thehematologic parameters during treatment with the BMP9 or BMP10polypeptide.

In certain embodiments, one or more hematologic parameters are measuredin patients who are being treated with a BMP9 or BMP10 polypeptide. Thehematologic parameters may be used to monitor the patient duringtreatment and permit adjustment or termination of the dosing with theBMP9 or BMP10 polypeptide or additional dosing with another therapeuticagent. For example, if administration of a BMP9 or BMP10 polypeptideresults in an increase in blood pressure, red blood cell level, orhemoglobin level, or a reduction in iron stores, then the dose of theBMP9 or BMP10 polypeptide may be reduced in amount or frequency in orderto decrease the effects of the BMP9 or BMP10 polypeptide on the one ormore hematologic parameters. If administration or a BMP9 or BMP10polypeptide results in a change in one or more hematologic parametersthat is adverse to the patient, then the dosing of the BMP9 or BMP10polypeptide may be terminated either temporarily, until the hematologicparameter(s) return to an acceptable level, or permanently. Similarly,if one or more hematologic parameters are not brought within anacceptable range after reducing the dose or frequency of administrationof the BMP9 or BMP10 polypeptide then the dosing may be terminated. Asan alternative, or in addition to, reducing or terminating the dosingwith the BMP9 or BMP10 polypeptide, the patient may be dosed with anadditional therapeutic agent that addresses the undesirable level in thehematologic parameter(s), such as, for example, a blood pressurelowering agent or an iron supplement. For example, if a patient beingtreated with a BMP9 or BMP10 polypeptide has elevated blood pressure,then dosing with the BMP9 or BMP10 polypeptide may continue at the samelevel and a blood pressure lowering agent is added to the treatmentregimen, dosing with the BMP9 or BMP10 polypeptide may be reduce (e.g.,in amount and/or frequency) and a blood pressure lowering agent is addedto the treatment regimen, or dosing with the BMP9 or BMP10 polypeptidemay be terminated and the patient may be treated with a blood pressurelowering agent.

4. Pharmaceutical Preparations

In certain embodiments, BMP9 or BMP10 polypeptides of the presentinvention are formulated with a pharmaceutically acceptable carrier. Forexample, a BMP9 or BMP10 polypeptide can be administered alone or as acomponent of a pharmaceutical preparation. The subject compounds may beformulated for administration in any convenient way for use in human orveterinary medicine. As noted above, it may be desirable to prepare aBMP9 or BMP10 polypeptide in a formulation comprising a prodomainpolypeptide.

In certain embodiments, the therapeutic method of the invention includesadministering the preparation systemically, or locally as an implant ordevice. When administered, the pharmaceutical preparation for use inthis invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Therapeutically useful agents other than the BMP9 orBMP10 polypeptides which may also optionally be included in thepreparation as described above, may be administered simultaneously orsequentially with the subject BMP9 or BMP10 polypeptides.

Typically, compounds will be administered parenterally. Pharmaceuticalpreparations suitable for parenteral administration may comprise one ormore BMP9 or BMP10 polypeptides in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powders(e.g., lyophilates) which may be reconstituted into sterile injectablesolutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, sugars, ethanol, polyols(such as glycerol, propylene glycol, polyethylene glycol, and the like),and suitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate.

Further, the preparation may be encapsulated or injected in a form fordelivery to a target tissue site. In certain embodiments, preparationsof the present invention may include a matrix capable of delivering oneor more therapeutic compounds (e.g., BMP9 or BMP10 polypeptides) to atarget tissue site, providing a structure for the developing tissue andoptimally capable of being resorbed into the body. For example, thematrix may provide slow release of the BMP9 or BMP10 polypeptides. Suchmatrices may be formed of materials presently in use for other implantedmedical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the BMP9 or BMP10 polypeptides. The various factors include, but arenot limited to, the patient's red blood cell count, hemoglobin level,systolic or diastolic blood pressure or other diagnostic assessments,the desired target red blood cell count, the patient's age, sex, anddiet, the severity of any disease that may be contributing to adepressed red blood cell level, time of administration, and otherclinical factors. The addition of other known growth factors to thefinal composition may also affect the dosage. Progress can be monitoredby periodic assessment of red blood cell and hemoglobin levels, as wellas assessments of reticulocyte levels and other indicators of thehematopoietic process.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of BMP9 or BMP10 polypeptides. Such therapywould achieve its therapeutic effect by introduction of the BMP9 orBMP10 polynucleotide sequences into cells or tissues having thedisorders as listed above. Delivery of BMP9 or BMP10 polynucleotidesequences can be achieved using a recombinant expression vector such asa chimeric virus or a colloidal dispersion system. Preferred fortherapeutic delivery of BMP9 or BMP10 polynucleotide sequences is theuse of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or an RNA virus suchas a retrovirus. The retroviral vector may be a derivative of a murineor avian retrovirus. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). Anumber of additional retroviral vectors can incorporate multiple genes.All of these vectors can transfer or incorporate a gene for a selectablemarker so that transduced cells can be identified and generated.Retroviral vectors can be made target-specific by attaching, forexample, a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody. Those of skill in the art willrecognize that specific polynucleotide sequences can be inserted intothe retroviral genome or attached to a viral envelope to allow targetspecific delivery of the retroviral vector containing the BMP9 or BMP10polynucleotide.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of a BMP9 or BMP10 Polypeptide

BMP9 or BMP10 may be purchased from a commercial supplier, such as R&DSystems (Minneapolis, Minn.). Alternatively, a protocol such as thefollowing may be followed:

A human BMP-9 (bBMP9) cDNA construct was generated by replacing thenative signal sequence of BMP-9 with the signal sequence of tissueplasminogen activator (tPA) or another signal sequence. Examples ofleader sequences:

(i) Honey bee melittin (HBML): (SEQ ID NO: 14) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 15)MDAMKRGLCCVLLLCGAVFVSP

The DNA sequence encoding the tPA signal sequence was fused in-framewith the DNA sequence encoding the propeptide/mature region of BMP-9.This cDNA sequence was cloned into the pAID4 vector to encode a proteinwith the following unprocessed sequence:

(SEQ ID NO: 16)   1MDAMKRGLCC VLLLCGAVFV SPGAKPLQSW GRGSAGGNAH SPLGVPGGGL  51PEHTFNLKMF LENVKVDFLR SLNLSGVPSQ DKTRVEPPQY MIDLYNRYTS 101DKSTTPASNI VRSFSMEDAI SITATEDFPF QKHILLFNIS IPRHEQITRA 151ELRLYVSCQN HVDPSHDLKG SVVIYDVLDG TDAWDSATET KTFLVSQDIQ 201DEGWETLEVS SAVKRWVRSD STKSKNKLEV TVESHRKGCD TLDISVPPGS 251RNLPFFVVES NDHSSGTKET RLELREMISH EQESVLKKLS KDGSTEAGES 301SHEEDTDGHV AAGSTLARRK RSAGAGSHCQ KTSLRVNFED IGWDSWIIAP 351KEYEAYECKG GCFFPLADDV TPTKHAIVQT LVHLKFPTKV GKACCVPTKL 401SPISVLYKDD MGVPTLKYHY EGMSVAECGC RA BMP10 polypeptide expression cassette may be similarly produced:

(SEQ. ID. NO: 17)   1MDAMKRGLCC VLLLCGAVFV SPGASPIMNL EQSPLEEDMS LFGDVFSEQD GVDFNTLLQS  61MKDEFLKTLN LSDIPTQDSA KVDPPEYMLE LYNKFATDRT SMPSANIIRS FKNEDLFSQP 121VSFNGLRKYP LLFNVSIPHH EEVIMAELRL YTLVQRDRMI YDGVDRKITI FEVLESKGDN 181EGERNMLVLV SGEIYGTNSE WETFDVIDAI RRWQKSGSST HQLEVHIESK HDEAEDASSG 241RLEIDTSAQN KHNPLLIVFS DDQSSDKERK EELNEMISHE QLPELDNLGL DSFSSGPGEE 301ALLQMRSNII YDSTARIRRN AKGNYCKRTP LYIDFKEIGW DSWIIAPPGY EAYECRGVCN 361YPLAEHLTPT KHAIIQALVH LKNSQKASKA CCVPTKLEPI SILYLDKGVV TYKFKYEGMA 421VSECGCR

BMP-9 constructs were be transfected into a CHO DUKX B11 cell line thathas been engineered to express a soluble (secreted) form of PACE(Furin)(Genbank No. P09958). Co-expression of PACE facilitatespropeptide cleavage of BMPs. Clones were selected in 10 nM methotrexate(MTX) followed by amplification in 50 nM MTX to increase expression. Ahigh expressing clone was identified by dilution cloning and adapted toserum-free suspension growth to generate conditioned media forpurification. Optionally, a ubiquitous chromatin opening element (UCOE)may be included in the vector to facilitate expression. See, e.g.,Cytotechnology. 2002 January; 38(1-3):43-6.

Affinity purification was achieved by passage over an affinity columnprepared by loading and cross-linking a protein A column (MAbSelectSure, GE Healthcare Life Sciences) with an altered ActRIIb-Fcfusion protein having the following sequence:

(SEQ ID NO: 18)   1ETRECIYYNA NWELERTNQS GLERCEGEQD KRLHCYASWR NSSGTIELVK  51KGCWDDDFNC YDRQECVATE ENPQVYFCCC EGNFCNERFT HLPEAGGPEV 101TYEPPPTGGG THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV 151VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD 201WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ 251VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV 301DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKBMP-9 protein was eluted from the column with 0.1M glycine, pH 3.0.BMP10 may be prepared in the same manner. In the event that BMP9 orBMP10 is purified as a mixed solution of covalent and non-covalentdimers, the covalent and non-covalent forms may be separated using areverse phase HPLC, such as a Vydac C4 column eluted with a gradient of0 to 100% acetonitrile in the presence of 0.1% trifluoroacetic acid.Covalent/non-covalent dimer content may be assessed by comparison ofreducing and non-reducing SDS-PAGE.

Example 2. Administration of BMP9 to Wild-type Mice

To explore the effects of BMP9 on erythropoiesis, 10 C57BL/6 mice wererandomized (2 groups, 5 animals per group) to receive two doses ofvehicle control (TBS containing 0.8 mM HCl and 0.1% BSA), or BMP9 (10mg/kg) once daily for two days by intraperitoneal injection. Bloodsamples were taken via tail vein on the study termination date. At 48hours post treatment whole blood was obtained to determine completeblood counts (CBCs).

BMP9 increased RBC number, Hemoglobin (HGB) level and Hematocrit (HCT)by 32%, 34% and 31%, respectively, compared to vehicle controlsuggesting that BMP9 treatments results in increased red blood cells.There were no substantial effects on white blood cells or other bloodparameters.

This study demonstrated that BMP9 has a profound, selective and rapideffect in increasing levels of red blood cells in the bloodstream, asmeasured by erythrocyte count, hemoglobin level and hematocrit.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A method for increasing red blood cell levels or treating anemia associated with thalassemia in a patient, the method comprising administering to a patient in need thereof a polypeptide selected from the group consisting of a BMP9 polypeptide and a BMP10 polypeptide.
 2. The method of claim 1, wherein the polypeptide is a BMP9 polypeptide.
 3. The method of claim 2, wherein the BMP9 polypeptide comprises an amino acid sequence that is at least 63% identical to the sequence of SEQ ID NO.:
 3. 4. The method of claim 2, wherein the BMP9 polypeptide comprises an amino acid sequence that is encoded by a nucleic acid that hybridizes under stringent hybridization conditions to the a nucleic acid that is complementary to the sequence of nucleotides 1121-1450 of SEQ ID NO.:
 11. 5. The method of claim 2, wherein the BMP9 polypeptide comprises an amino acid sequence that is identical to the sequence of SEQ ID NO.:
 3. 6. The method of claim 1, wherein the polypeptide is a BMP10 polypeptide.
 7. The method of claim 6, wherein the BMP10 polypeptide comprises an amino acid sequence that is at least 63% identical to the sequence of SEQ ID NO.:
 6. 8. The method of claim 6, wherein the BMP10 polypeptide comprises an amino acid sequence that is encoded by a nucleic acid that hybridizes under stringent hybridization conditions to the a nucleic acid that is complementary to the sequence of nucleotides 1108-1431 of SEQ ID NO.:
 12. 9. The method of claim 6, wherein the BMP10 polypeptide comprises an amino acid sequence that is identical to the sequence of SEQ ID NO.:
 6. 10. The method of claim 1, wherein the BMP9 polypeptide or BMP10 polypeptide is administered in a pharmaceutical preparation.
 11. The method of claim 10, wherein the pharmaceutical preparation comprises a prodomain polypeptide selected from the group consisting of a BMP9 prodomain polypeptide and a BMP10 prodomain polypeptide.
 12. The method of claim 11, wherein the prodomain polypeptide is a BMP9 prodomain polypeptide.
 13. The method of claim 12, wherein the prodomain polypeptide comprises an amino acid sequence that is at least 80% identical to the sequence of amino acids 23-319 of SEQ ID NO.:
 1. 14. The method of claim 12, wherein the prodomain polypeptide comprises the amino acid sequence of amino acids 23-319 of SEQ ID NO.:
 1. 15. The method of claim 12, wherein the pharmaceutical preparation comprises a BMP9 polypeptide noncovalently associated with the BMP9 prodomain polypeptide.
 16. The method of claim 12, wherein the pharmaceutical preparation comprises a BMP10 polypeptide noncovalently associated with the BMP9 prodomain polypeptide.
 17. The method of claim 11, wherein the prodomain polypeptide is a BMP10 prodomain polypeptide.
 18. The method of claim 17, wherein the prodomain polypeptide comprises an amino acid sequence that is at least 80% identical to the sequence of amino acids 22-316 of SEQ ID NO.:
 4. 19. The method of claim 17, wherein the prodomain polypeptide comprises the amino acid sequence of amino acids 22-316 of SEQ ID NO.:
 4. 20-39. (canceled) 